Comparison of the effects of 7.2% hypertonic saline and 20% mannitol on whole blood coagulation and platelet function in dogs with suspected intracranial hypertension - a pilot study

Osmotherapy is commonly used in the treatment of intracranial hypertension (ICH) due to a variety of causes, including head trauma, intracranial neoplasia, infection or hemorrhage, and status epilepticus [1]. The principle goal of osmotherapy is to shift fluid from the intracellular into the extracellular compartment using intravenous hyperosmolar agents, thereby reducing brain edema and improving cerebral perfusion pressure [2]. Although 10–20% mannitol is considered the gold standard hyperosmolar agent in the treatment of ICH [3], mannitol-induced osmotic diuresis may cause hypovolemia and reduction in cerebral perfusion pressure [1]. In recent years, 3.0–7.5% hypertonic saline (HTS) has gained popularity in the treatment of ICH [4, 5] as it has less pronounced diuretic effects and therefore does not cause hypovolemia [1, 6]. Indeed, in the face of hypovolemic shock and traumatic brain injury, HTS provides the advantage of volume expansion, restoring adequate cerebral perfusion pressures, and reducing brain edema, which makes it superior to mannitol in trauma patients with shock [7, 8].

Both mannitol and HTS have been shown to interfere with whole blood coagulation and platelet function [9]. This is in part due to dilutional coagulopathy. Furthermore, 7.2% HTS may directly disturb both fibrin formation and platelet function [10], and mannitol may interfere with coagulation by reducing clot strength [11]. In addition, hyperosmolarity is supposed to lead to impairment of both whole blood coagulation and platelet function [9, 12]. In consequence, the safety of using these agents in patients with ICH and intracranial hemorrhage remains unclear [4, 11, 13]. Previous in vitro studies in humans have demonstrated anticoagulant effects of both mannitol and HTS [9, 14], although one clinical study failed to demonstrate any negative effect on hemostasis using either solution in patients undergoing elective intracranial surgery [15]. Similarly, in vitro studies in dogs demonstrated that both mannitol and HTS have negative effects on coagulation in a dose-dependent fashion, although in vivo studies in dogs in a clinical setting are lacking [16, 17]. As a result, current guidelines for osmotherapy in dogs with ICH are largely extrapolated from experimental data and human literature [18, 19].

The aim of this study was to compare the effects of clinically recommended doses of 20% mannitol and 7.2% HTS on whole blood coagulation using rotational thromboelastometry (ROTEM®) and on platelet function using a platelet function analyzer (PFA®) in dogs with suspected ICH. Based on a previous canine in vitro study, we expected that mannitol would have a greater impact on coagulation than HTS [17]. A second aim was to compare plasma osmolarity after either osmotherapeutic solution. Knowledge of the extent to which these solutions may impact global hemostasis may influence clinical decision-making in the selection of solutions for individual dogs.

The study was designed as a prospective, randomized, non-blinded cohort study using client-owned dogs with suspected ICH. The trial was approved the Animal Experiment Committee of the Swiss Federal Veterinary Office (registration number BE 90/13), and informed owner consent was obtained for all dogs.

A total of 30 dogs were included in the study (15 dogs in each group). Dogs had a mean age of 6.2 ± 3.4 years and a mean body weight of 25.9 ± 13.6 kg. There were 13 female dogs (5 sexually intact, 8 spayed) and 17 male dogs (8 sexually intact, 9 castrated). No significant difference was found between groups for age (P = 0.99), body weight (P = 0.31), or gender (P = 0.72). Breeds represented were mixed-breed (n = 4), French bulldog (n = 4), golden retriever (n = 4), Labrador retriever (n = 3), border collie (n = 2) and 1 each of boxer, cocker spaniel, flat coated retriever, fox terrier, German shepherd, great Dane, Jack Russel terrier, Keeshond, Malinois, mudi, Saint Bernard, Tervuren, and white Swiss shepherd. Of the 30 dogs, 24 underwent magnet resonance imaging and 1 dog underwent computed tomography. Underlying diseases were intracranial neoplasia (n = 8, mannitol group; n = 8, HTS group), intoxication (n = 3, mannitol group; n = 1, HTS group), head trauma (n = 2, mannitol group; n = 1, HTS group), meningoencephalitis (n = 2, mannitol group), hydrocephalus internus (n = 1, HTS group), presumptive hypertensive encephalopathy (n = 1, HTS group), and undetermined in the remaining 3 dogs (HTS group).

The present study evaluated the effects on coagulation of intravenous 20% mannitol and 7.2% HTS in a cohort of dogs with suspected ICH using ROTEM® and PFA® analyses. Only minimal differences in coagulation parameters were found between dogs treated with mannitol and those receiving HTS at currently recommended doses. Indeed, a significant difference between the groups in percent of values relative to baseline (T₀) was only found for FIBTEM® CT, which showed a shorter time until clot detection at T₅ and T₆₀ in dogs receiving HTS. This was mirrored by a shorter FIBTEM® CT at T₅, T₆₀, and T₁₂₀ compared to T₀ in the HTS group. Furthermore, a short-lived decrease in EXTEM® and FIBTEM® MCF was observed after mannitol. However, median ROTEM® values remained within institutional RIs at all time points in both groups. In contrast, PFA® values increased above institutional RIs after mannitol (T₅) and HTS (T₅ and T₆₀), although no significant differences were found between groups or time points. In terms of the importance of the detected changes, alterations in FIBTEM® CT are most likely of no relevance, as in human medicine only the clot firmness variables (A5, A10, etc., and MCF) of the FIBTEM® assay are used (eg., for deciding on the replacement of fibrinogen sources) [25]. The very mild changes in MCF and Ct_PFA may be signs for a mannitol induced, short-lived fibrin polymerization defect (decrease in MCF FIBTEM®) as well as short lived platelet-fibrin interaction defect, such as impairment of GPIIb/IIIa receptor mediated binding (decrease in MCF EXTEM® combined with impaired platelet function) which might become more prominent after higher doses. Furthermore, plasma osmolarity was significantly increased by 15–25 mOsm/L for up to 1 h (mannitol group) and up to 2 h (HTS group), respectively, but no difference between the groups was detected.

Previous in vitro studies in humans and dogs indicate that both mannitol and HTS affect primary and secondary hemostasis in a dose-dependent fashion by delayed clot formation and impairment of fibrin clot firmness [12, 14, 16, 17]. Indeed, a 1:22 dilution of whole blood with 7.2% HTS (mimicking a dose of 4 ml/kg) significantly affected Ct_PFA and ROTEM® EXTEM® CFT and MCF in dogs [16]. Moreover, 20% mannitol affected Ct_PFA and ROTEM® EXTEM® variables to a greater extent than equimolar 3% HTS in dilutions mimicking recommended clinical doses [17]. However, given the in vitro nature of these studies and absence of the endothelium and compensatory mechanisms, such as buffering, electrolyte homeostasis, and metabolic degradation and excretion of the drug, results may not reflect in vivo conditions [26]. Moreover, in vitro dilution of blood may result in a more significant dilution of coagulation factors than the corresponding in vivo dose, impacting the kinetics of clot formation. Finally, effects of transendothelial fluid shifts, which are a crucial effect of osmotherapy, are essentially eliminated by in vitro studies.

The findings of the present study only partially confirm recent in vitro findings [17]. The less pronounced effect of mannitol in the present study may to some extent be due to disparate osmolarities of the HTS solutions evaluated (3% HTS in the previous in vitro study compared to 7.2% in the present study). Indeed, hyperosmotic stress may result in impaired enzymatic function in the clotting cascade, slower clot formation and a weaker clot [9, 10, 12]. In the present study no significant difference in plasma osmolarity was found between the two groups despite the higher osmolarity and more rapid administration of HTS. Likewise, no significant difference in plasma osmolarity was found between 7.2% NaCl/HES 200/0.5 and 15% mannitol in a previous study in 40 adult neurosurgical patients [27]. This may, in part, explain the lack of difference in ROTEM® and PFA® parameters between the two osmotherapeutic groups. However, the extent to which the changes observed in ROTEM® and PFA® parameters were due to increased osmolarity, increased sodium load, or to additional hemostatic disturbances from each hyperosmolar molecule itself remains unclear. Lastly, the volume administered in the present study was lower than dilutions used in some previous in vitro studies, which may have masked potential coagulation impairing effects at higher doses.

Similar to the current findings, in two recent studies in people undergoing elective craniotomy or suffering from traumatic brain injury, respectively, no difference in standard coagulation tests and ROTEM® analysis was found between patients administered 20% mannitol and those receiving 3% HTS [15, 28].

To the authors’ knowledge, the present study is the first investigation evaluating platelet function with PFA® after intravenous administration of mannitol or HTS in dogs. Despite the 30% increase in Ct_PFA after mannitol at T₅ and some PFA values above the RI in both groups, the changes did not reach statistical significance and returned to normal within the two-hour study period. A clear advantage of one of the osmotherapeutics to avert platelet dysfunction was therefore not evident, although mannitol may have more pronounced but shorter lived effects compared to HTS.

In contrast to the aforementioned in vitro studies [16, 17], dogs in the present study were not healthy as they all were affected by conditions causing suspected ICH. Of the included dogs, 53% had intracranial neoplasia, albeit equally distributed between both treatment groups. Furthermore, 5 dogs (n = 3, HTS group; n = 2, mannitol group) included in the study had received one dose of glucocorticoids (≤1 mg/kg) within 7 days prior to the study, which may be expected to increase clot strength and decrease clot lysis in thromboelastography [29]. However, effects on thromboelastographic amplitude and clot lysis in the respective study were found after two 2 and 4 days of treatment, respectively, with an immunosuppressive dose of prednisone (median dose 2.07 mg/kg per 24 h). In contrast, the dogs in the present study received prednisolone in a lower dose and only once. Nevertheless, given heterogeneous diseases and previous treatments, some dogs included in this study were expected to have some abnormal initial PFA® or ROTEM® measurements. Indeed, MCF values were above the RI in EXTEM® (n = 1) and FIBTEM® (n = 5). This confounder was cancelled out in the evaluation of differences between time points by using the percentage of parameters relative to those measured at T₀ instead of absolute values, allowing evaluation of a cohort that truly represents the target population of dogs receiving osmotherapy. Given the small numbers of dogs affected by different diseases, no analyses of associations between disease and coagulation parameters was performed and further studies are needed to establish whether certain conditions leading the ICH are associated with coagulation abnormalities in dogs.

The current guidelines for viscoelastic coagulation testing recommend blood sampling for ROTEM® by atraumatic puncture of the jugular vein [30]. However, as compression of the jugular vein is not recommended in dogs with ICH, samples were taken from the lateral saphenous veins in the present study [18]. The extent to which this may have affected results is not clear.

Artefactual hypercoagulability using ROTEM® analysis has been shown to occur due to low red blood cell mass [31, 32]. Likewise, low hematocrits and low platelet count was found to prolong PFA® results [23]. In the present study, some dogs were slightly anemic following administration of osmotherapeutics but hematocrits did not fall below 0.30 L/L and platelet counts not fall below 100 × 10⁹/L in any dog at any time point, which is within the current recommendations for accurate PFA® testing [23].

The current study has potential limitations. Firstly, simultaneous standard coagulation testing (prothrombin time (PT), partial thromboplastin time (aPTT), and fibrinogen concentration) was not assessed. Although ROTEM® analysis enables dynamic assessment of whole blood coagulation and platelet function, standard coagulation testing may have helped in the interpretation of the clinical relevance of findings. However, both PT and aPTT are essentially limited to answering the question whether any intervention exerts effects on thrombin generation, depending on the coagulation factors reflected in the respective assay. As we were not expecting the interventions in our study to exert impacts on thrombin generation, the information obtainable by additional PT and aPTT assays would be limited to detect potential effects induced by dilution. Furthermore, both poor and good associations between standard coagulation tests and ROTEM® analyses have been reported [33, 34], and information about platelet function, fibrin polymerization, and platelet interaction with fibrin is not provided by plasmatic coagulation tests. Further, although there are no results of any conventional fibrinogen assay available, variables of clot firmness (MCF and A10) of ROTEM® FIBTEM® assays are highly correlated with the fibrinogen concentration [31] and adequately provide information about potential fibrin-polymerization defects that are not detectable using standard lab tests. Even if potentially elevated fibrinogen concentrations in some of the dogs might have alleviated coagulation impairment, importantly no relevant alteration of fibrin polymerization was found after osmotherapy in the present study.

Another limitation was that some dogs received additional crystalloid fluid therapy during the study period as treatment was largely at the discretion of the clinicians. Previous in vitro studies in dogs demonstrated a dilutional coagulopathy caused by 0.9% saline on viscoelastic coagulation and PFA® testing [16, 35]. Contrariwise, a recent in vivo study in healthy anesthetized dogs administered with a 15 ml/kg-bolus of isotonic buffered crystalloids did not led to relevant ROTEM® and PFA® abnormalities [36]. Nevertheless, simultaneous crystalloid fluid therapy might have impacted results in individual dogs in the present study. Lastly, although the previous in vitro study evaluated a 3% HTS solution, dogs in the present study were given a 7.2% HTS solution as this was the established institutional treatment protocol for dogs with ICH. It is likely that more significant differences between groups would have been found had mannitol been compared with 3% HTS instead of 7.2% HTS.

In conclusion, results of this pilot study suggest that mannitol and HTS do not differ in their effects on coagulation when administered in the currently recommended doses in dogs with suspected ICH. Moreover, no clinically relevant impairment of whole blood coagulation was found following treatment with either solution, whereas a short-lived impairment of platelet function was found after both solutions. Further studies are warranted before recommendations can be made with regards to treatment of individual animals.